The long-term objective of this application is to understand the molecular mechanisms that regulate axon- glial interactions of myelination, potentially revealing targets of new therapeutic strategies to aid in remyelination. This proposal is focused on examining how Necl-4, a cell adhesion molecule found to mediate axon-glial interactions along the internode, promotes myelination through interactions with the Par-3/aPKC polarity complex. Necl-4, expressed by myelinating Schwann cells, was recently shown to be essential for the myelination of axons in the peripheral nervous system. A key question left unanswered is how Necl-4 plays a critical role in myelination. A molecule that is a candidate to interact with Necl-4 is Partitioning-defective 3 (Par- 3), of the Par-aPKC polarity complex. The absence of Par-3 in Schwann cells phenocopies the effects seen in Necl-4 knockdown cells, suggesting that Par-3 may be the downstream effector of Necl-4. Our preliminary results indicate that Necl-4, through its PDZ-binding domain, interacts with Par-3 directly. Necl-4 also interacts with members of the 4.1 family of cytoskeletal proteins via its FERM-binding domain. Together these findings suggest a model where Par-3 is recruited by Necl-4 to the adaxonal membrane of the Schwann cell upon axon-glial contact. The localization of Par-3 to this site in turn may target and activate aPKC/Par-6, generating anterior-posterior polarity and the formation of a leading edge in the Schwann cell that drives spiral wrapping of the axon. This Necl-4/Par complex is then stabilized through interactions between Necl-4 and protein 4.1G and its association with spectrin and the actin cytoskeleton, thereby promoting long-term viability of the myelin sheath. Understanding the functional role of the Necl-4 complex could yield new insight into the molecular mechanisms that occur during axon-glial contact and myelination of axons. Three specific aims are proposed in order to characterize the role of the Necl-4/Par-3 complex and Necl- 4/4.1G interaction in promoting PNS myelination. The first aim is to determine if the PDZ- binding domain of Necl-4 is required for myelination by recruiting Par-3 to the adaxonal membrane using a knockdown/rescue strategy with a modified Necl-4 construct. The second aim will address whether the localization of Par-3 to the adaxonal membrane in turn recruits aPKC/Par-6 and activates aPKC, triggering signaling pathways that drive Schwann cell myelination. The last aim will characterize mice deficient in 4.1G to determine the role of 4.1G in stabilizing the Necl-4/Par complex and the long-term effects on myelinated fiber integrity. PUBLIC HEALTH RELEVANCE: Narrative Myelin-related disorders affects millions of people worldwide and with no cure currently available, represent a significant impact to public health. The goal of this study is to understand the proteins that regulate axon-glial interactions and formation of myelin. Elucidating the mechanisms of myelination could reveal new insights into the pathophysiology of myelin-related disorders and provide therapeutic targets.